Structural Basis for Induced Formation of the Inflammatory Mediator

Structural basis for induced formation of the inflammatory mediator prostaglandin E2 Caroline Jegerscho¨ ld*†, Sven-Christian Pawelzik‡, Pasi Purhonen*, Priyaranjan Bhakat*§, Karina Roxana Gheorghe*, Nobuhiko Gyobu¶, Kaoru Mitsuokaʈ, Ralf Morgenstern**, Per-Johan Jakobsson†‡, and Hans Hebert*† *Department of Biosciences and Nutrition, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, Novum, S-141 57 Huddinge, Sweden; ‡Department of Medicine, Rheumatology unit and Karolinska Biomic Center and **Institute of Environmental Medicine, Karolinska Insitutet, S-171 77 Stockholm, Sweden; and ¶Japan Biological Information Research Center, Japan Biological Informatics Consortium, and ʈBiological Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi 2-41-6, Koto-Ku, Tokyo 135-0064, Japan Edited by Richard Henderson, Medical Research Council, Cambridge, United Kingdom, and approved May 7, 2008 (received for review March 23, 2008) Prostaglandins (PG) are bioactive lipids produced from arachidonic protein expression in this context seems resistant to anti-TNF␣ acid via the action of cyclooxygenases and terminal PG synthases. treatment or oral administration of corticosteroids (12, 13). Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an Together, these findings suggest that MPGES1 is a potential inducible glutathione-dependent integral membrane protein that target for development of therapeutic agents for the treatment catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into of several diseases (10). PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and Results pain, and is therefore regarded as a primary target for develop- Structural Determination. We have determined the structure of ment of novel antiinflammatory drugs. To provide a structural human MPGES1 in complex with the tripeptide ␥-L-glutamyl- basis for insight in the catalytic mechanism, we determined the L-cysteinyl-glycine, glutathione (GSH) at 3.5 Å in-plane resolu- structure of MPGES1 in complex with glutathione by electron tion using electron crystallography (Table S1). The protein, crystallography from 2D crystals induced in the presence of phos- fused to an N-terminal His6-tag, was overexpressed in Esche- pholipids. Together with results from site-directed mutagenesis richia coli and purified in a Triton X-100 solubilized form. 2D and activity measurements, we can thereby demonstrate the role crystals were grown by dialysis in the presence of added phos- of specific amino acid residues. Glutathione is found to bind in a pholipids at a low lipid to protein ratio. The crystals often U-shaped conformation at the interface between subunits in the appeared as rounded rectangular sheets extending several ␮min protein trimer. It is exposed to a site facing the lipid bilayer, which the long dimension and Ϸ1 ␮m across (Fig. S2A). In the forms the specific environment for the oxidoreduction of PGH2 to crystalline sheets, the protein molecules were tightly packed and PGE2 after displacement of the cytoplasmic half of the N-terminal tilted 20° relative to the normal of the layer (Fig. S3). This transmembrane helix. Hence, insight into the dynamic behavior of arrangement explained an earlier observation that no end on MPGES1 and homologous membrane proteins in inflammation and views revealing the positions of transmembrane helices were detoxification is provided. observed from 0° projection maps (Fig. S2A) as had been demonstrated earlier for the MAPEG members MGST1 (14, 15) electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein and LTC4S (16). Polar interactions within one unit cell involving arginine residues with exposed side chains at either the lumenal icrosomal prostaglandin E synthase 1 (MPGES1) is the or the cytoplasmic face of the protein were found to play Mkey enzyme in pathology related production of PGE2 from pertinent roles for crystal contacts (Fig. S3). cyclooxygenase (Cox) derived PGH2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxy- Overall Structure. The subunits of MPGES1 form a homotrimer genase activating protein (FLAP), leukotriene C4 synthase (Fig. 1) in a similar way as for the other structurally characterized (LTC4S), microsomal glutathione transferase (MGST)1, MAPEG members, MGST1 (17), FLAP (18), and LTC4S (19, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES 20) and in agreement with earlier low resolution and hydrody- known and catalyzes the oxidoreduction of prostaglandin endo- namic data on MPGES1 (5). Our result thus supports the Ϫ1 Ϫ1 peroxide H2 into PGE2 with an apparent kcat/Km of 310 mM s suggestion that a trimeric arrangement is common to all [supporting information (SI) Fig. S1]. The enzyme equally well MAPEG proteins (21). Because the 2D crystals of MPGES1 are catalyses the oxidoreduction of endocannabinoids into prosta- orthorhombic the symmetry component within the trimer is a glandin glycerol esters (4) and PGG2 into 15-hydroperoxy-PGE2 local pseudo threefold axis. The variations in the immediate (5). In addition, the enzyme confers low glutathione transferase surroundings of the subunits in the asymmetric units of the unit and glutathione-dependent peroxidase activities (5). The biological significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1. Author contributions: C.J., P.-J.J., and H.H. designed research; C.J., S.-C.P., P.P., P.B., K.R.G., N.G., K.M., and H.H. performed research; C.J., S.-C.P., P.P., P.B., R.M., P.-J.J., and H.H. MPGES1 protein expression levels are in most cases low, and analyzed data; and C.J., R.M., P.-J.J., and H.H. wrote the paper. proinflammatory stimuli induce its cellular expression and ac- The authors declare no conflict of interest. tivity, which is prevented by corticosteroids (1, 6–8). The This article is a PNAS Direct Submission. predominant source of PGH2 seems derived from Cox-2, al- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, though Cox-1 may also contribute (9). Studies, mainly from www.pdb.org (PDB ID code 3DWW). disruption of the MPGES1 gene in mice, indicate key roles for †To whom correspondence may be addressed. E-mail: [email protected], caroline. MPGES1-generated PGE2 in pathological conditions such as [email protected], or [email protected]. chronic inflammation, pain, fever, anorexia, atherosclerosis, §Present address: Molecular and Health Technologies, Commonwealth Scientific and stroke and tumorigenesis (10). Recently, a role for MPGES1 in Industrial Research Organization, 343 Royal Parade, Parkville VIC 3052, Australia. regulating neonatal respiration was described in ref. 11. This article contains supporting information online at www.pnas.org/cgi/content/full/ MPGES1 has been shown to be overexpressed in rheumatic 0802894105/DCSupplemental. diseases such as rheumatoid arthritis and myositis and the © 2008 by The National Academy of Sciences of the USA 11110–11115 ͉ PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802894105 Downloaded by guest on September 27, 2021 Fig. 2. Intra- and intersubunit interactions in MPGES1. (A) Stabilization of the cytoplasmic face of transmembrane helices 2 and 3. (B) Interactions between the same helices close to the GSH binding site. (C) Stabilization of the trimer through the His-72–Glu-77 ion pair. inner core structure, which narrows down into complete closure toward the lumenal side by aromatic side chains. The funnel shaped opening toward the opposite cytoplasmic side is partly covered by the three large connections between TM1s and TM2s. These domains protrude toward the loops between TM3 and TM4 of neighboring subunits. Fig. 1. Overall structure of MPGES1. (A)2Fo Ϫ Fc map contoured at 1.2 ␴ as Essentially no charged side chains are found exposed toward seen from the side at the interface between two subunits. (B and C) The trimer the phospholipid bilayer for MPGES1 (Fig. S5). The side chain subunits have been colored in ribbon representation. (B) Top view from the of Lys-26 in the center of TM1 points toward the interior of the face corresponding to the cytoplasm when the protein is positioned in its structure and makes an ion pair with Asp-75 of TM2 in the same natural membrane environment. (C) Side view with the cytoplasmic side up. subunit. This interaction gives rise to a distortion and bending of The transmembrane helices of one subunit have been labeled at their C- TM1. Arg-110, located in the center of TM3, is not exposed to terminal ends. the lipid bilayer but rather involved in intermolecular contacts. Compared with MPGES1, the crystal structure of LTC4S has cell suggest local structural variations at least in the peripheral more exposed charged side chains (Fig. S5). parts of the trimers. Mutagenesis. Our present structure and effects of site-directed The His6-tagged N terminus located on the lumenal side of the membrane embedded protein is flexible and the determined mutagenesis on activity have identified several important intra- and intermolecular contacts (Fig. 2 and Table 1). The cytoplas- model starts at Pro-11. The model begins with a long transmem- BIOCHEMISTRY mic face of TM2 is crucial for GSH binding and is stabilized brane (TM) helix ending at Lys-41. It is followed by a relatively toward TM3 of the same subunit close to its loop connection to large cytoplasmic loop region. Sequence alignment of MAPEG TM4 (Fig. 2A). The polar side chain residues involved in this members (Fig. S4) shows that this domain is highly variable and interaction Glu-66, Arg-67, Arg-70, and Tyr-117 are highly it is thus unlikely to be crucial for the catalytic functions of the conserved in MAPEG (Fig. S4). Among the human members MAPEG enzymes. The second TM helix, similar in length as the MGST3 has substitutions to asparagine and cysteine at positions first one, spans residues 61–90.

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